Abstract

Mutations in the dystrophin gene cause Duchenne muscular dystrophy and result in the loss of dystrophin and the entire dystrophin-glycoprotein complex (DGC) from the sarcolemma. We show that sarcospan (SSPN), a unique tetraspanin-like component of the DGC, ameliorates muscular dystrophy in dystrophin-deficient mdx mice. SSPN stabilizes the sarcolemma by increasing levels of the utrophin-glycoprotein complex (UGC) at the extrasynaptic membrane to compensate for the loss of dystrophin. Utrophin is normally restricted to the neuromuscular junction, where it replaces dystrophin to form a functionally analogous complex. SSPN directly interacts with the UGC and functions to stabilize utrophin protein without increasing utrophin transcription. These findings reveal the importance of protein stability in the prevention of muscular dystrophy and may impact the future design of therapeutics for muscular dystrophies.

SSPN regulates UGC levels by interacting directly with this complex. (A) Skeletal muscle from mdx and SSPN-Tg:mdx tissue was solubilized in modified radioimmunoprecipitation assay buffer, and 60-μg protein samples were resolved by SDS-PAGE and immunoblotted using antibodies against dystrophin (Dys), utrophin (Utrn), DGs (α- and β-DG), exogenous SSPN (hSSPN), and SGs (α-, β-, and γ-SG). As expected, exogenous hSSPN was only detected in SSPN-Tg:mdx muscle. Expression levels of DGs and SGs were dramatically elevated in SSPN-Tg:mdx muscle samples. Utrophin, a homologue of dystrophin, is up-regulated in SSPN-Tg:mdx tissue. Equal loading of mdx and SSPN-Tg:mdx lysates was confirmed by Coomassie blue (CB) staining. Molecular masses of individual proteins are indicated in parentheses. (B) Quantitative RT-PCR analysis was used to determine the level of utrophin mRNA in both SSPN-Tg:mdx and mdx tissue. Data are normalized to GAPDH controls and are represented relative to control values (100%). Utrophin mRNA levels were not statistically different (standard t test; P = 0.258) between SSPN-Tg:mdx and mdx controls, suggesting that SSPN-Tg expression did not affect utrophin transcription. Values represent mean ± SEM (error bars). (C) SSPN is a core component of the UGC in SSPN-Tg:mdx mice. Purified UGC proteins were separated by ultracentrifugation through 5–30% sucrose gradients. Fraction 1 represents the lightest region of the gradient. Fractions were analyzed by immunoblotting with antibodies to utrophin (Utrn), DGs (α- and β-DG), SGs (α-, and γ-SG), and exogenous SSPN (hSSPN) as indicated. SSPN and utrophin are enriched in fraction 4, demonstrating that SSPN is a core component of the UGC. The molecular mass of each component is identical to that indicated in A.